Among the many pathways controlling cell proliferation and differentiation, genes of the retinoblastoma protein (pRb) regulatory network stand out as frequent, if not obligatory, targets for mutation or deregulation during human tumorigenesis. Although biochemical, tissue culture, and transgenic studies in mice have implicated pRb family members in a wide range of activities, the full spectrum of authentic pRb functions during normal development has yet to be resolved. In addition, new studies suggest that pRb may exert its anti-oncogenic effects through a variety of distinct mechanisms, although the critical functions of pRb in tumor suppression remain to be determined. Our long-term objectives are to understand the molecular, cellular, and developmental functions of pRb family proteins and to mechanistically link the pRb pathway to other cellular networks. To this end, we have used a range of genetic and molecular methods to characterize novel functions of lin-35, the sole Rb ortholog in C. elegans, including roles in organ morphogenesis, intestinal homeostasis, and cell fate determination. In addition, using transcriptome profiling, we have identified many LIN-35 target genes that rep- resent novel candidate cell cycle regulators. Our main objectives fall into three categories. In Aim 1, we will fo- cus on further elucidating the LIN-35 regulatory network controlling pharyngeal morphogenesis. Specifically, we will explore the roles of several new genes that we have implicated as functioning within this pathway and will identify additional components through established screening methods. In Aim 2, we will study the roles of LIN-35 and SLR-2, a Zn-finger protein, in regulating intestinal function and gene expression. This will be accomplished using forward genetics and molecular approaches and will include directed studies to examine the functional connection between LIN-35 and genes identified in a genome-wide RNAi suppressor screen. In Aim 3, we will follow up on microarray and bioinformatical analyses to identify novel cell cycle components. Functions for candidate genes will be determined using a variety of sensitized genetic backgrounds and in vivo as- says, which will allow us to determine roles for these genes in cell cycle progression and regulation. The successful completion of these aims will enhance our basic understanding of both cell cycle and non-cell cycle functions for pRb family members and will provide mechanistic insights into the roles of LIN-35 and associated pathways in controlling the formation and function of two principal organs involved in nutrient uptake and utilization. This proposed research will also support efforts to define authentic in vivo activities for pRb family members and to identify genetic modifiers that may augment or diminish the phenotypic effects of Rb mutations. As such, these studies have strong relevance to understanding the tumor-suppressing functions of pRb and the many cellular factors that comprise the greater pRb regulatory network in humans. Our published findings and extensive preliminary results provide a solid foundation and logical framework for carrying out the proposed experiments. PUBLIC HEALTH RELEVANCE: The goal of our research is to understand the biological functions of the Retinoblastoma protein (pRb), which normally protects against tumor formation in humans. Because a protein that is closely related to pRb is present in the nematode model organism C. elegans, we will use this powerful system to study the functions of pRb family proteins in order to learn more about their roles during normal development. This will lead to a better understanding of the anti-cancer activities carried out by pRb in humans and may suggest new approaches for anti-cancer therapies.